Human disease syndromes involving cystic nephropathies and sensori-neural deficits are linked to defects of cilia, for example, Senior-Loken syndrome, Bardet-Biedl Syndrome, Juvenile Nephronophthisis. Cilia in their stationary or motile forms serve both sensory and effector functions in cellular physiology. Genetic and biochemical analysis of ciliogenesis in simple organisms such as Chlamydomonas and C.elegans have led to detailed understanding of cellular processes invoved in cilia formation, however their significance in pathophysiology of ciliary disease necessitates studies in vertebrates. In characterization of the zebrafish cystic mutant fleer we identified widespread cilia defects that arise from reduction in glutamylation of axonemal tubulins. Polyglutamylation is a posttranslational modification detected on tubulins and functionally implicated in cellular processes such as axon extension, centriole stability. Fleer contains tetratricopeptide repeats (TPRs) but lacks tubulin tyrosine ligase (TTLL) motifs, a defining feature of polyglutamylase enzyme complex subunits.The C.elegans Fleer homolog DYF-1 translocates within the cilia. TPR motifs are implicated in protein interactions and we hypothesize that Fleer regulates ciliary transport of tubulin glutamylase by scaffolding an association between TTLL subunit(s) and kinesins. This proposal seeks to define the role of Fleer in biochemical process required for normal cilia formation. Specifically we will 1) assess whether Fleer exists in protein complexes together with TTLL and/or kinesin subunits and what protein subdomain(s) are required for cilia localization and protein interactions. 2) Identify novel protein interactions of Fleer using tandem affinity purification technique and yeast two hybrid assay and 3) Conditionally abrogate Fleer function in transgenic zebrafish expressing dominant negative Fleer. Successful completion of proposed studies will identify important components of the Fleer regulated network of proteins that modulate tubulin glutamylation. Components altered in the conditional knockdown model will identify key proteins involved in progression of ciliopathies that could serve as therapeutic targets. Expression analysis of different TTLL proteins in the prelimnary studies will also yield important information on how distinct tubulin modification codes are specified and provide scope for my independent career pathway.
Cilia and basal bodies play important roles in vertebrate development and physiology, as sensors of mechanical, chemical and osmotic stimuli. Human disease syndromes involving cystic nephropathies and sensori-neural deficits are linked to defects of cilia. Success of the proposed research will allow us to identify specific, tubulin-glutamylation dependent processes associated with ciliopathies that may underlie human disease conditions.
|Pathak, Narendra; Austin-Tse, Christina A; Liu, Yan et al. (2014) Cytoplasmic carboxypeptidase 5 regulates tubulin glutamylation and zebrafish cilia formation and function. Mol Biol Cell 25:1836-44|
|Pathak, Narendra; Austin, Christina A; Drummond, Iain A (2011) Tubulin tyrosine ligase-like genes ttll3 and ttll6 maintain zebrafish cilia structure and motility. J Biol Chem 286:11685-95|
|Pathak, Narendra H; Drummond, Iain A (2009) Polyglutamylation and the fleer gene. Methods Cell Biol 94:317-32|